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Design, Synthesis, Antibacterial Activity, and Molecular Docking Studies of Novel Hybrid 1,3-Thiazine-1,3,5-Triazine Derivatives as Potential Bacterial Translation Inhibitor

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Design, Synthesis, Antibacterial Activity, and Molecular Docking Studies of Novel Hybrid 1,3-Thiazine-1,3,5-Triazine Derivatives as Potential Bacterial Translation Inhibitor
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  Design, Synthesis, Antibacterial Activity, andMolecular Docking Studies of Novel Hybrid 1,3-Thiazine-1,3,5-Triazine Derivatives as PotentialBacterial Translation Inhibitor Udaya P. Singh 1 *, Manish Pathak 1 ,Vaibhav Dubey 1 , Hans R. Bhat 1 , PrashantGahtori 2 and Ramendra K. Singh 3 1 Department of Pharmaceutical Sciences, Sam Higginbottom Institute of Agriculture Technology and Sciences, Formerly Allahabad Agricultural Institute, Deemed to be University, 211007 Allahabad, India  2  Faculty of Pharmacy, Uttarakhand Technical University, Dehradun,Uttarakhand 248007, India  3  Nucleic Acids Research Laboratory, Department of Chemistry,University of Allahabad, 211002 Allahabad, India *Corresponding author: Udaya P. Singh, udaysingh98@gmail.com  Some novel hybrid 1,3-thiazine-1,3,5-triazine deriv-atives were synthesized and tested for antibacte-rial activity. Compounds 8c and 8f were foundactive against Gram positive and Gram negativemicroorganisms. Molecular docking studies havebeen performed on eubacterial ribosomal decod-ing A site ( Escherichia coli   16S rRNA A site) torationalize the probable mode of action, bindingaffinity, and orientation of the molecules at theactive site of receptor. The structures of all thesenewly synthesized compounds were confirmed bytheir elemental analyses and spectral data tech-niques viz. IR,  1 H NMR,  13 C NMR, and mass.Key words:  1,3,5-Triazine, 1,3-thiazine, antibacterial, docking, trans-lation inhibitionReceived 1 February 2012, revised 16 April 2012 and accepted for publi-cation 5 June 2012 Antibiotic resistance is all the time more recognized as a seriousand permanent public health concern and is usually considered tobe a consequence of wide use and misuse of antibiotics. Surveil-lance data for  Streptococcus pneumoniae  , a common cause of bac-terial respiratory tract infections, disclosed that 24% of isolateswere not susceptible to penicillin. Moreover, resistance to severalother antibacterial drugs is common; of which 1.5% of isolateswere resistant to cefotaxime (a third generation cephalosporin), andresistance to the newer fluoroquinolone antimicrobials has alreadybeen reported (1). In Europe, it is estimated that at least 25 000people die each year from infections because of antibiotic-resistantbacteria, which also result in around 2.5 million extra hospital days(2). As a result, increased incidence of bacterial resistance to cur-rently available antibiotics altogether increases healthcare costassociated with it. According to recent study by Robert and cowork-ers, it has been calculated that annual costs of antibiotic-resistantinfections to the U.S. Healthcare system comes to be in excess of$20 billion (3).In an our effort to design effective antimicrobial compound derivedfrom 1,3,5-triazine, we had earlier reported a novel heterocyclichybrid skeleton encompassing thiazole and 1,3,5-triazine connectedthrough –NH– linker (4,5). Structure-activity relationship suggestedthat, the thiazole amine pendant is well tolerated along with thepresence of electron withdrawing groups on phenyl amine on eitherside of 1,3,5-triazine core. Prompted by the results, we furtherdevised a series of analogues as potential antimicrobials by keepingthiazole fragment rigid and having diverse substitution pattern onboth wings of 1,3,5-triazine by amine and aromatic, aliphatic frag-ments tethered via amine and mercapto (-S-) bridge (6). Resultsrevealed that analogues having amine bridge were more effectivethan their respective mercapto equivalents and further explicatedthe critical structural requirement of electron withdrawing groups.Over the past decade, bacterial ribosome is a key target for natu-rally occurring antibiotics, including the macrolides, tetracyclines,chloramphenicol, aminoglycosides, and the newly discovered syn-thetic oxazolidinones as well (7). Recently, 3,5-diamino-piperidinyltriazines (DAPT) having 1,3,5-triazine core scaffold, identified as anovel class of antibacterial agent have been found active in murinemodel, that target the bacterial decoding-site RNA  in vitro   and inhi-bit bacterial growth by a translation-dependent mechanism. Thesederivatives are considered to be as a mimetics of the natural ami-noglycoside antibiotics (8–10). Nevertheless, facts drawn throughmolinspiration program in our earlier studies allowed us to reportnuclear receptor binding domain for antibacterial action of 1,3,5-tri-azine derivatives (11).Prompted by these findings, present study deals with the synthesisof hybrid analogues of 1,3-thiazine-1,3,5-triazine as core skeletontethered through –NH– linkage and its antibacterial activity deter-mination. To define binding mode of these constitutive compoundsas plausible bacterial translation inhibitor, molecular docking studies 1 Chem Biol Drug Des 2012  Research Article ª  2012 John Wiley & Sons A/S  doi: 10.1111/j.1747-0285.2012.01430.x  were carried out on eubacterial ribosomal decoding A site ( Escheri- chia coli   16S rRNA A site) receptor domain. The rationale behindthe present study was to optimize the substituent on pendant posi-tion, therefore, we expand the ring of earlier optimized substitutedphenyl thiazole-2-amine fragment by one carbon atom and introduc-tion of another substituted phenyl ring resulting in 4,6-substiututedphenyl-6 H  -1,3-thiazin-2-amine. Experimental Material and method  Melting points of compounds were determined in open capillarytubes Hicon melting point apparatus and are uncorrected. Thin layerchromatographic analysis was done to monitor the completion ofreaction as well as for identification and characterization of com-pounds. The different mobile phases were selected according to theassumed polarity of the products. The spots were visualized byexposure to iodine vapor and UV light. The structures of the inter-mediate compounds were established on the basis of spectral (FT-IR,  1 H NMR, mass) and elemental analysis, whereas structures oftitle hybrid analogues on the basis of FT-IR,  1 H NMR,  13 C NMR,mass spectral, and elemental analysis data. FT-IR (in 2.0 per cm,flat, smooth, abex, KBr) spectra were recorded on Biored FTs spec-trophotometer.  1 H NMR spectra were recorded on Bruker Avance II400 and 300 MHz NMR spectrometer in CDCl 3 -d 6  and DMSO usingtetramethylsilane (TMS) as internal standard.  13 C NMR spectrawere recorded on Bruker Avance II 300 NMR Spectrometer. Chemi-cal shifts are reported in parts per million (ppm,  d ) and signals aredescribed as s (singlet), d (doublet), t (triplet), q (quartet), and m(multiplet). The FAB mass spectrum was recorded on a THERMOFinnigan LCQ Advantage max ion trap mass spectrometer, sampleswere introduced into ESI source through Finnigan surveyor autosampler. The mobile phase MeOH   ⁄   CAN:H 2 O (90:10) flowed at a rateof 250  l L   ⁄   min by MS pump. Ion spray voltage was set at 5.3 KVand capillary voltage 34 V. The scan run upto 2.5 min and the spec-tra averaged over 10 scan at peak top in TLC. Elemental analysiswas carried out on a Vario EL III CHNOS elemental analyzer. Chemistry  The synthesis of title analogues were carried out through followingsteps. Step 1: Synthesis of 4,6-substituted di-phenyl1,3-thiazine-2-amine General procedure for synthesis of substituted chalcone derivatives 3(a–f).  Dissolved substituted acetophenone,  1  (0.01 mol) andsubstituted benzaldehyde,  2  (0.01 mol) in 50 mL ethanolic solutionand stirred for 30 min, followed by dropwise addition of aqueoussodium hydroxide (0.05 mol), further stirring continued for 24 h.After completion of the reaction as reaction monitored by TLC usingthe mobile phase as  n  -butanol:acetic acid:water (4:3:1), a crudeproduct was obtained as substituted chalcone,  3 ( a – f ). The resultantsolid was then filtered off, washed with water, dried, and re-crys-tallized from ethanol. 3-(2-Nitrophenyl)-1-phenylprop-2-en-1-one (3a).  Yellow crystals;Yield: 76%; mp: 123–125   C; R f   : 0.56; FT-IR ( m max ; per cm KBr):1654 (C=O), 1592 (C=C), 1548.28–1446.06 (aromatic C=N);  1 H NMR(400 MHz, CDCl 3 -d 6 , TMS):  d  7.99–7.98 (m, 2H, H ¢ 2  H ¢ 6 ), 7.56–7.53(m, 3H, H ¢ 3  H ¢ 4  H ¢ 5 ), 7.52 (d, 1H,  J   = 8.4 Hz H 6 ), 7.51 (m, 3H, H 3  H 4 H 5 ), 6.89 (d, 1H,  J   = 15.3 Hz H b ), 6.38 (d, 1H,  J   = 15.3 Hz H a ); Anal.Calcd. For C 15 H 11 NO 3 : C, 71.14; H, 4.38; N, 5.53. Found: C, 71.12;H, 4.39; N, 5.58. 3-(2-Nitrophenyl)-1-(4-nitrophenyl)prop-2-en-1-one (3b).  Light Yellowcrystals; Yield: 85%; mp: 133–134   C; R f   : 0.64; FT-IR ( m max ; per cmKBr): 1656 (C=O), 1594 (C=C), 1548.28–1446.06 (aromatic C=N);  1 HNMR (400 MHz, CDCl 3 -d 6 , TMS):  d  7.97–7.96 (m, 2H, H ¢ 2  H ¢ 6 ),7.54–7.53 (m, 2H, H ¢ 3  H ¢ 5 ), 7.51 (d, 1H,  J   = 8.3 Hz H 6 ), 7.51 (m, 3H,H 3  H 4  H 5 ), 6.89 (d, 1H,  J   = 15.3 Hz H b ), 6.38 (d, 1H,  J   = 15.3 HzH a ); Anal. Calcd. For C 15 H 10 N 2 O 5 : C, 60.41; H, 3.38; N, 9.39. Found:C, 60.32; H, 3.36; N, 9.40. 1-(4-Hydroxyphenyl)-3-(2-nitrophenyl)prop-2-en-1-one (3c).  Brown crys-tals; Yield: 83%; mp: 129–130   C; R f   : 0.54; FT-IR ( m max ; per cm KBr):1659 (C=O), 1595 (C=C), 1546.28–1448.06 (aromatic C=N);  1 H NMR(400 MHz, CDCl 3 -d 6 , TMS)  d  ppm: 7.97–7.98 (m, 2H, H ¢ 2  H ¢ 6 ), 6.94–6.96 (m, 2H, H ¢ 3  H ¢ 5 ), 5.28 (s, 1H, Ar-OH), 7.51 (d, 1H,  J   = 8.2 HzH 6 ), 7.50 (m, 3H, H 3  H 4  H 5 ), 6.89 (d, 1H,  J   = 15.3 Hz H b ), 6.38 (d,1H,  J   = 15.3 Hz H a ); Anal. Calcd. For C 15 H 11 NO 4 : C, 66.91; H, 4.12;N, 5.20. Found: C, 66.90; H, 4.14; N, 5.20. 3-(2-Chlorophenyl)-1-(4-nitrophenyl)prop-2-en-1-one (3d).  Dark Yel-low crystals; Yield: 87%; mp: 142–143   C; R f   : 0.68; FT-IR ( m max ; percm KBr): 1652 (C=O), 1590 (C=C), 1546–1448 (aromatic C=N), 810,754;  1 H NMR (400 MHz, CDCl 3 -d 6 , TMS)  d  ppm: 7.97–7.98 (m, 2H,H ¢ 2  H ¢ 6 ), 7.53–7.51 (m, 2H, H ¢ 3  H ¢ 5 ), 7.48 (d, 1H,  J   = 8.3 Hz H 6 ),7.48–749 (m, 3H, H 3  H 4  H 5 ), 6.89 (d, 1H,  J   = 15.3 Hz H b ), 6.37 (d,1H,  J   = 15.3 Hz H a ); Anal. Calcd. For C 15 H 10 ClNO 3 : C, 62.62; H,3.50; N, 4.87. Found: C, 62.65; H, 3.53; N, 4.86. 1-(4-Chlorophenyl)-3-(4-nitrophenyl)prop-2-en-1-one (3e).  Brown yellowcrystals; Yield: 79%; mp: 138–139   C; R f   : 0.47; FT-IR ( m max ; per cmKBr): 1658 (C=O), 1594 (C=C), 1543–1441 (aromatic C=N), 1230, 880; 1 H NMR (400 MHz, CDCl 3 -d 6 , TMS)  d  ppm: 7.95–7.96 (m, 2H, H ¢ 2 H ¢ 6 ), 7.54–7.53 (m, 2H, H ¢ 3  H ¢ 5 ), 7.51 (m, 3H, H 3  H 4  H 5 ), 6.89 (d, 1H, J   = 15.3 Hz H b ), 6.38 (d, 1H,  J   = 15.3 Hz H a ); Anal. Calcd. ForC 15 H 10 ClNO 3 : C, 62.62; H, 3.50; N, 4.87. Found: C, 62.66; H, 3.52;N, 4.88. 1,3-Bis(4-nitrophenyl)prop-2-en-1-one (3f).  Orange crystals; Yield:64%; mp: 153–154   C; R f   : 0.78; FT-IR ( m max ; per cm KBr): 1654(C=O), 1596 (C=C), 1545–1443 (aromatic C=N), 1240, 880, 749;  1 HNMR (400 MHz, CDCl 3 -d 6 , TMS)  d  ppm: 7.97–7.98 (m, 2H, H ¢ 2  H ¢ 6 ),7.53–7.52 (m, 2H, H ¢ 3  H ¢ 5 ), 8.01–8.03 (m, 2H, H 2 ,H 6 ), 8.17 (m, 2H,H 3  H 5 ), 6.88 (d, 1H,  J   = 15.3 Hz H b ), 6.37 (d, 1H,  J   = 15.3 Hz H a );Anal. Calcd. For C 15 H 10 N 2 O 5 : C, 60.41; H, 3.38; N, 9.39. Found: C,60.43; H, 3.38; N, 9.38. General procedure for synthesis of substituted 1,3-thiazine from cor- responding chalcone derivative 3(a–f).  The corresponding substi-tuted chalcone derivatives  3 ( a – f ) (0.01 mol) and thiourea (0.01 mol)was dissolved in ethanol (50 mL) and refluxed the mixture, while a Singh et al. 2  Chem Biol Drug Des   2012  solution of potassium hydroxide (0.05 mol) in water (10 mL) addedportion-wise for 2 h. Then again refluxed it for further 6 h and afterthat resultant mixture was poured into ice-cold water. A solid prod-uct  4 ( a – f ) thus obtained was filtered off, dried and crystallizedfrom ethanol. 6-(2-Nitrophenyl)-4-phenyl-6H-1,3-thiazin-2-amine (4a).  Yellow crys-tals; Yield: 78%; mp: 163–164   C; R f   : 0.45; FT-IR ( m max ; per cm KBr):3356 (NH),1687 (C-N), 1596 (C=C), 1656–1645 (aromatic C=N), 1522,1351 (Ar-NO 2 ), 876, 756;  1 H NMR (400 MHz, CDCl 3 -d 6 , TMS)  d  ppm:5.63 (s, 1H, H5), 7.33–7.30 (m, 3H), 7.66 (d, 1H,  J   = 7.9 Ar-H), 8.35 (d,1H,  J   = 7.1, 5.43 Ar-H), 5.52 (bs, 1H); Anal. Calcd. For C 16 H 13 N 3 O 2 S:C, 61.72; H 4.21; N, 13.50. Found: C, 61.66; H, 4.23; N, 13.52. 6-(2-Nitrophenyl)-4-(4-nitrophenyl)-6H-1,3-thiazin-2-amine (4b).  Browncrystals; Yield: 67%; mp: 169–171   C; R f   : 0.65; FT-IR ( m max ; per cmKBr): 3359 (NH),1683 (C-N), 1599 (C=C), 1656–1645 (aromatic C=N),1524, 1353 (Ar-NO 2 ), 876, 756;  1 H NMR (400 MHz, CDCl 3 -d 6 , TMS) d  ppm: 5.68 (s, 1H, H5) 7.43–7.40 (m, 3H), 7.69 (d, 1H,  J   = 7.8 Ar-H), 8.06 (d, 1H,  J   = 7.1, 5.4 Ar-H), 5.56 (bs, 1H); Anal. Calcd. ForC 16 H 12 N 4 O 4 S: C, 53.93; H 3.39; N, 15.72. Found: C, 53.90; H, 3.39;N, 15.71. 4-(2-Amino-6-(2-nitrophenyl)-6H-1,3-thiazin-4-yl)phenol (4c).  BrownYellow crystals; Yield: 69%; mp: 176–178   C; R f   : 0.54; FT-IR ( m max ;per cm KBr): 3359 (NH),1686 (C-N), 1600 (C=C), 1646–1635 (aro-matic C=N), 1521, 1353 (Ar-NO 2 ), 889, 778;  1 H NMR (400 MHz,CDCl 3 -d 6 , TMS)  d  ppm: 5.46 (s, 1H, H5) 7.37–7.67 (m, 3H), 7.69 (d,1H,  J   = 7.6 Ar-H), 8.06 (d, 1H,  J   = 7.1, 5.4 Ar-H), 5.45 (bs, 1H);Anal. Calcd. For C 16 H 13 N 3 O 3 S: C, 58.70; H 4.00; N, 12.84. Found: C,57.45; H, 4.12; N, 12.85. 6-(2-Chlorophenyl)-4-(4-nitrophenyl)-6H-1,3-thiazin-2-amine (4d). Light Yellow crystals; Yield: 84%; mp: 183–184   C; R f   : 0.65; FT-IR( m max ; per cm KBr): 3359 (NH),1686 (C-N), 1634 (C=C), 1648–1630(aromatic C=N), 1520, 1353 (Ar-NO 2 ), 890, 772;  1 H NMR (400 MHz,CDCl 3 -d 6 , TMS)  d  ppm: 5.73 (s, 1H, H5) 7.31–7.38 (m, 3H), 7.62 (d,1H,  J   = 7.9 Ar-H), 8.09 (d, 1H,  J   = 7.2, 5.3 Ar-H), 5.45 (bs, 1H);Anal. Calcd. For C 16 H 12 ClN 3 O 2 S: C, 55.57; H 3.50; N, 12.15. Found:C, 55.51; H, 3.55; N, 12.07. 4-(4-Chlorophenyl)-6-(4-nitrophenyl)-6H-1,3-thiazin-2-amine (4e). Dark Brown crystals; Yield: 76%; mp: 189–192   C; R f   : 0.58; FT-IR( m max ; per cm KBr): 3354 (NH),1683 (C-N), 1631 (C=C), 1648–1630(aromatic C=N), 1524, 1350 (Ar-NO 2 ), 880, 782;  1 H NMR (400 MHz,CDCl 3 -d 6 , TMS)  d  ppm: 5.52 (s, 1H, H5) 7.35–7.36 (m, 3H), 7.56 (d,1H,  J   = 7.6 Ar-H), 8.06 (d, 1H,  J   = 7.3, 5.3 Ar-H), 5.56 (bs, 1H);Anal. Calcd. For C 16 H 12 ClN 3 O 2 S: C, 55.57; H 3.50; N, 12.15. Found:C, 55.51; H, 3.55; N, 12.07. 4,6-Bis(4-nitrophenyl)-6H-1,3-thiazin-2-amine (4f).  Orange crystals;Yield: 79%; mp: 196–198   C; R f   : 0.57; FT-IR ( m max ; per cm KBr):3359 (NH),1682 (C-N), 1635 (C=C), 1648–1630 (aromatic C=N),1524, 1353 (Ar-NO 2 ), 890, 756;  1 H NMR (400 MHz, CDCl 3 -d 6 ,TMS):  d  5.76 (s, 1H, H5) 7.34–7.36 (m, 3H), 7.64 (d, 1H,  J   = 7.6Ar-H), 8.10 (d, 1H,  J   = 7.3, 5.2 Ar-H), 5.52 (bs, 1H); Anal. Calcd.For C 16 H 12 N 4 O 4 S: C, 53.93; H 3.39; N, 15.72. Found: C, 53.91; H,3.40; N, 15.72. Step 2: Procedure for synthesis of 6-chloro- N  2 , N  4 -bis(3-nitrophenyl)-1,3,5-triazine-2,4-diamine (7) 3-Nitro aniline  6  (0.2 mol) was added into 100 mL of acetone attemperature 40–45   C. The solution of 2,4,6-trichloro-1,3,5-triazine( 5 ) (0.1 mol) in 25 mL acetone was added slowly, stirred the reac-tion for 3 h followed by drop-wise addition of NaHCO 3  solution(0.1 mol) taking care that reaction mixture does not become acidic.Completion of the reaction was analyzed by TLC utilizing benzene:ethyl acetate as mobile phase (9:1). The product was filtered andwashed with cold water and re-crystallized with ethanol to affordpure compound 6-chloro- N  2 , N  4 -bis(3-nitro phenyl)-1,3,5-triazine-2,4-diamine,  7 .Brownish black crystals; Yield: 86%; mp: 143–145   C; MW: 387.74;R f   : 0.55; FT-IR ( m max ; per cm KBr): 3289.56 (N–H secondary), 3055.70(C–H broad), 1548.28–1446.06 (aromatic C=N);  1 H NMR (400 MHz,CDCl 3 -d 6 , TMS):  d  7.40 (t, 4H, 4 = CH-aromatic), d 7.32(t, 4H,4 = CH aromatic), 3.62 (d, 2H, 2-NH aromatic); Elemental analysisfor C 15 H 10 ClN 7 O 4 : Calculated: C, 46.46; H, 2.60; N, 25.29. Found: C,46.48; H, 2.65; N, 25.26. Step 3: General procedure for synthesis ofhybrid 1,3-thiazine-1,3,5-triazine analogues 8(a–f) 6-Chloro- N  2 , N  4 -bis(3-nitrophenyl)-1,3,5-triazine-2,4-diamine ( 7 ) (0.1 mol)was added into 50 mL of 1,4-dioxane at temperature 40–45   C. Asolution of substituted 1,3-thiazine  4 ( a – f ) (0.1 mol) in 35 mL 1,4-diox-ane was added slowly to above solution and stirred for 90 min fol-lowed by drop-wise addition of K 2 CO 3  (0.1 mol), re-fluxed the reactionmixture at 135–145   C for 9 h. The product was filtered and washedwith cold water and re-crystallized with ethanol to afford the corre-sponding pure products  8 ( a – f ). N  2 , N  4 -Bis(3-nitrophenyl)- N  6 -(6-(2-nitrophenyl)-4-phenyl-6H-1,3-thiazin-2-yl)-1,3,5-triazine-2,4,6-triamine ( 8a ). Black crystals; Yield: 86%;mp: 145–147   C; R f   : 0.56; FT-IR ( m max ; per cm KBr): 3278 (N-H stretch ,-NH 2 ), 2927 (C-H stretch , Aromatic), 1577 Aromatic (C=C ring stretch ),1529 (-C=N ring stretch ), 1349 (-NO 2stretch ), 1127 (C-N stretch ), 834 (N-H deformation ), 1403–1092 Aromatic (-C-H in plane deformation ), 737 Aro-matic (-C-H out of plane deformation ), 1238 (C-S stretch );  1 H NMR(400 MHz, CDCl 3 -d 6 , TMS):  d  3.90 (bs, 3H, NH), 6.91 (d, 1H, J   = 9.8Hz, Ar-H), 6.92–6.98 (d, 1H,  J   = 9.9Hz, Ar-H), 7.26 (s, 1H, thi-azine), 7.42–7.44 (d, 1H,  J   = 8.4Hz, Ar-H), 7.63–7.99 (m, 1H, Ar-H),7.73 (d, 1H,  J   = 8.7Hz, Ar-H), 7.99–8.01(d, 1H,  J   = 4.8Hz, 1H, Ar-H),8.39–8.42 (d, 1H,  J   = 8.4Hz, Ar-H), 8.66 (S, 1H, Ar-H);  13 C NMR(400 MHz, CDCl 3 ):  d  170.42, 133.95, 114.91, 77.65, 77.23, 76.80;ESI-MS (m   ⁄   z): 525.10(M + H + ); Anal. Calcd. For C 31 H 22 N 10 O 6 S: C,56.19; H, 3.35; N, 21.14. Found: C, 55.12; H, 3.41; N, 21.12. N  2 , N  4 -Bis(3-nitrophenyl)- N  6 -(6-(2-nitrophenyl)-4-(4-nitrophenyl)-6H-1,3-thiazin-2-yl)-1,3,5-triazine-2,4,6-triamine ( 8b ). Yellow crystals; Yield:78%; mp: 169–171   C; R f   : 0.51; FT-IR ( m max ; per cm KBr): 3276 (N-H stretch , -NH 2 ), 2366 (C-H stretch , Aromatic), 1585–1433 Aromatic (C=Cring stretch ), 1613 (-C=N ring stretch ), 1348 (-NO 2stretch ), 1317 (C-S stretch ),1299 (C-N stretch ), 994 (N-H deformation ), 1401-1090 Aromatic (-C-H in planedeformation ), 700 Aromatic (-C-H out of plane deformation ), 1317 (C-S stretch ); Design, Synthesis, Antibacterial Activity, and Molecular Docking Studies of 1,3,5-triazines Chem Biol Drug Des   2012  3  1 H NMR (400 MHz, CDCl 3 -d 6 , TMS): 4.07 (bs, 3H, NH), 7.03 (d, 1H, J   = 8.1Hz, Ar-H), 7.15 (d, 1H,  J   = 6Hz, Ar-H), 7.27 (s, 1H, thiazine),7.73 (d, 1H,  J   = 7.5Hz, Ar-H), 7.95–7.97 (d, 1H,  J   = 8.4Hz, Ar-H), 7.79(d, 1H,  J   = 7.8Hz, Ar-H), 8.00 (d, 1H,  J   = 7.2Hz, 1H, Ar-H), 8.07–8.62(m, 1H, Ar-H), 8.62 (S, 1H, Ar-H);  13 C NMR (400 MHz, CDCl 3 ):  d 129.84, 126.45, 77.43, 77.00, 76.58, 29.67; ESI-MS (m   ⁄   z):424.17(M + H + ); Anal. Calcd. For C 31 H 21 N 11 O 8 S: C, 52.62; H, 2.99; N,21.77. Found: C, 52.60; H, 3.01; N, 21.81. 4-(2-(4,6-Bis(3-nitrophenylamino)-1,3,5-triazin-2-ylamino)-6-(2-nitro- phenyl)-6H-1,3-thiazin-4-yl)phenol (8c).  Light Yellow crystals; Yield:76%; mp: 182-184   C; R f   : 0.46; FT-IR ( m max ; per cm KBr): 3383 (N-H stretch , -NH 2 ), 3567 (O-S stretch ) 2366 (C-H stretch , Aromatic), 1527 Aro-matic (C=C ring stretch ), 1570 (-C=N ring stretch ), 1400-1093 Aromatic (-C-H in plane deformation ), 1347 (-NO 2stretch ), 1296 (C-O stretch ), 1211 (C-S stretch ), 1168 (C-N stretch ), 835 (N-H deformation ), 753 Aromatic (-C-H outof plane deformation ), 670 Aromatic (Ar-Cl stretch );  1 H NMR (400 MHz,CDCl 3 -d 6 , TMS):  d  3.78 (bs, 3H, NH), 6.92-7.95 (d, 1H,  J   = 6.8Hz,Ar-H), 7.48–7.51 (d, 1H,  J   = 6.9Hz, Ar-H), 7.55–7.58 (d, 1H, J   = 6.3Hz, Ar-H), 8.18–8.20 (d, 1H,  J   = 6.6Hz, 1H, Ar-H), 8.20–8.42(d, 1H,  J   = 9.6Hz, Ar-H), 7.32 (s, 1H, thiazine), 7.66–7.97 (m, 1H, Ar-H), 6.92-6.95 (d, 1H,  J   = 6Hz, Ar-H), 8.66 (S, 1H,  J   = 5.5Hz, Ar-H); 13 C NMR (400 MHz, CDCl 3 ):  d  148.44, 133.54, 130.38, 129.33,124.85, 122.25, 77.43, 77.00, 76.58, 40.49, 40.21; ESI-MS (m   ⁄   z):657.19 (M + H + ); Anal. Calcd. For C 31 H 22 N 10 O 7 S: C, 54.86; H, 3.27;N, 20.64. Found: C, 55.05; H, 3.27; N, 20.65. N  2  -(6-(2-chlorophenyl)-4-(4-nitrophenyl)-6H-1,3-thiazin-2-yl)-N  4 , N  6 -bis(3-nitrophenyl)-1,3,5-triazine-2,4,6-triamine ( 8d ). Yellow crystals;Yield: 86%; mp: 174–176   C; R f   : 0.31; FT-IR ( m max ; per cm KBr):3385 (N-H stretch , -NH 2 ), 2927 (C-H stretch , Aromatic), 1599 (-C=N ring- stretch), 1528 Aromatic (C=C ringstretch ), 1400–1091 Aromatic (-C-H  in planedeformation ), 1354 (-NO 2stretch ), 1178 (C-N stretch ), 834 (N-H deformation ),738 Aromatic (Ar-Cl stretch ), 611 Aromatic (-C-H out of plane deformation ),572.50 (C-S stretch );  1 H NMR (400 MHz, CDCl 3 -d 6 , TMS):  d  3.57 (bs,3H, NH), 6.70 (s, 1H,  J   = 6.8Hz, Ar-H), 6.93–6.91 (d, 1H,  J   = 6Hz,Ar-H), 6.97 (d, 1H,  J   = 8.7Hz, Ar-H), 7.22 (s, 1H, thiazine), 7.38 (d,1H,  J   = 7.8Hz, Ar-H), 7.51 (d, 1H,  J   = 7.8Hz, Ar-H), 7.90-7.78 (m, 1H,Ar-H), 8.14 (d, 1H,  J   = 9Hz, 1H, Ar-H), 8.59 (s, 1H,  J   = 8.4Hz, Ar-H); 13 C NMR (300 MHz, CDCl 3 ):  d  148.60, 129.80, 129.16, 127.22,126.48, 123.86, 113.91, 77.43, 77.01, 76.58, 67.05, 30.29; ESI-MS(m   ⁄   z): 655.10(M + H + ); Anal. Calcd. For C 31 H 21 ClN 10 O 6 S: C, 53.41; H,3.04; N, 20.09. Found: C, 53.43; H, 3.03; N, 20.07. N  2 -(4-(4-Chlorophenyl)-6-(4-nitrophenyl)-6H-1,3-thiazin-2-yl)- N  4 , N  6 -bis(3-nitrophenyl)-1,3,5-triazine-2,4,6-triamine ( 8e ). Light Browncrystals; Yield: 83%; mp: 168-170   C; R f   : 0.52; FT-IR ( m max ; per cmKBr): 3385 (N-H stretch , -NH 2 ), 2924 (C-H stretch , Aromatic), 1522 (-C=Nring stretch ), 1431 Aromatic (C=C ring stretch ), 1402–1091 Aromatic (-C-H in plane deformation ), 1351 (-NO 2stretch ), 1238 (C-S stretch ), 1177 (C-N stretch ), 834 (N-H deformation ), 670 Aromatic (Ar-Cl stretch ), 612 Aromatic(-C-H out of plane deformation );  1 H NMR (400 MHz, CDCl 3 -d 6 , TMS):  d 3.98 (bs, 1H, NH), 6.93-6.91 (d, 1H,  J   = 6Hz, Ar-H), 7.13 (d, 1H, J   = 9.3Hz, Ar-H), 7.26 (s, 1H, thiazine), 7.50–7.53 (d, 1H,  J   = 6.0Hz,Ar-H), 7.62–7.80 (m, 1H, Ar-H),7.87 (d, 1H,  J   = 8.7Hz, Ar-H), 7.99–8.02 (d, 1H,  J   = 9.2Hz, Ar-H), 8.06–8.09 (d, 1H,  J   = 7.8Hz, Ar-H),8.24–8.28 (d, 1H,  J   = 8.1Hz, Ar-H);  13 C NMR (300 MHz, CDCl 3 ):  d 77.42, 77.00, 76.58, 67.05; ESI-MS (m   ⁄   z): 655.10 (M + H + ); Anal.Calcd. For C 31 H 21 ClN 10 O 6 S: C, 53.41; H, 3.04; N, 20.09. Found: C,53.45; H, 3.01; N, 20.08. N  2 -(4,6-Bis(4-nitrophenyl)-6H-1,3-thiazin-2-yl)- N  4 , N  6 -bis(3-nitrophenyl)-1,3,5-triazine-2,4,6-triamine ( 8f ). Yellow crystals; Yield: 68%; mp:154–156   C; R f   : 0.47; FT-IR ( m max ; per cm KBr): 3384 (N-H  stretch , -NH 2 ), 1522 Aromatic (C=C ring stretch ), 1399–1091 Aromatic (-C-H inplane deformation ), 1343 (-NO 2stretch ), 1239 (C-S stretch ), 1177 (C-N stretch ),994 (N-H deformation ) 801 (N-H rocking ), 670 Aromatic (-C-H out of planedeformation );  1 H NMR (400 MHz, CDCl 3 -d 6 , TMS):  d  3.76 (bs, 3H, NH),7.10-7.07 (d, 1H,  J   = 8.8Hz, Ar-H), 7.33 (s, 1H, thiazine), 7.66–7.67(d, 1H,  J   = 8.8Hz, Ar-H), 7.94–7.92 (d, 1H,  J   = 8.4Hz, Ar-H), 8.01–7.99 (d, 1H,  J   = 8.8Hz, Ar-H), 8.10–8.08 (d, 1H,  J   = 8.4Hz,Ar-H),8.15–8.13 (d, 1H,  J   = 7.2Hz, Ar-H), 8.72–8.35 (d, 1H,  J   = 8.8Hz, Ar-H), 8.20–8.18 (d, 1H,  J   = 8.8Hz, Ar-H);  13 C NMR (400 MHz, CDCl 3) : d  148.60, 129.80, 129.23, 127.22, 126.48, 123.86; ESI-MS (m   ⁄   z):424.17(M + H + ); Anal. Calcd. For C 31 H 21 N 11 O 8 S: C, 52.62; H, 2.99;N, 21.77. Found: C, 52.70; H, 3.02; N, 21.79. Molecular docking studies  The 3D X-ray crystal structure of paromomycin docked into the eu-bacterial ribosomal decoding A site ( E. coli   16S rRNA A site;1j7t:pdb) was used as starting model for this study. The proteinwas prepared, docked, scored, and the molecular dynamics simula-tion carried out using standard procedures. All computational analy-sis was carried out using Discovery Studio 2.5 (Accelrys SoftwareInc., San Diego; http://www.accelrys.com). Preparation of receptor The target protein that complexed with paramomycin (PDB ID: 1j7t)was taken, the ligand paramomycin extracted, and the bond orderwere corrected. The hydrogen atoms were added, and their posi-tions were optimized using the all-atom CHARMm (version c32b1)forcefield with Adopted Basis set Newton Raphson (ABNR) minimi-zation algorithm, until the root mean square (r.m.s) gradient forpotential energy was <0.05 kcal   ⁄   mol   ⁄    (18,19). Using the 'BindingSite' tool panel available in DS 2.5, the minimized eubacterial ribo-somal decoding A site ( E. coli   16S rRNA A site) was defined asreceptor, binding site was defined as volume occupied by the ligandin the receptor, and an input site sphere was defined over the bind-ing site with a radius of 5 . The center of the sphere was takento be the center of the binding site, and side chains of the residuesin the binding site within the radius of the sphere were assumedto be flexible during refinement of postdocking poses. The receptorhaving defined binding site was used for the docking studies. Ligand setup Using the built-and-edit module of DS 2.5, various ligands werebuilt all-atom CHARMm forcefield parameterization was assigned,and then minimized using the ABNR method. A conformationalsearch of the ligand was carried out using a stimulated annealingmolecular dynamics (MD) approach. The ligand was heated to atemperature of 700 K and then annealed to 200 K. Thirty suchcycles were carried out. The transformation obtained at the end ofeach cycle was further subjected to local energy minimization, using Singh et al. 4  Chem Biol Drug Des   2012  the ABNR method. The 30 energy-minimized structures were thensuperimposed and the lowest energy conformation occurring in themajor cluster was taken to be the most probable conformation. Docking and Scoring Molecular docking is a significant computational method used toforecast the binding of the ligand to the receptor binding site byvarying position and conformation of the ligand keeping the recep-tor rigid. LigandFit (20) protocol of DS 2.5 was used for the dockingof ligands with eubacterial ribosomal decoding A site (pdb id:1j7t) a . The LigandFit docking algorithm combines a shape compari-son filter with a Monte Carlo conformational search to generatedocked poses consistent with the binding site shape. These initialposes are further refined by rigid body minimization of the ligandwith respect to the grid based calculated interaction energy usingthe Dreiding forcefield (21). The receptor protein conformation waskept fixed during docking, and the docked poses were further mini-mized using all-atom CHARMm (version c32b1) forcefield and smartminimization method (steepest descent followed by conjugate gradi-ent), until r.m.s gradient for potential energy was <0.05 kcal   ⁄   mol   ⁄   .The atoms of ligand and the side chains of the residues of thereceptor within 5  from the center of the binding site were keptflexible during minimization. The description of ligand scoring (-PLP1, -PLP2, -PMF, Lig_Internal_Energy, Binding Energy, and DockScore) were given earlier in result and discussion section. Further-more, determination of binding energy to assess the binding affinityof ligands for receptor was calculated by employing highest stableligand-receptor complex through the protocol 'Calculate BindingEnergies' within DS 2.5 using the default settings. Antibacterial screening  Minimum inhibitory concentration Entire hybrid compounds were screened for their minimum inhibitoryconcentration (MIC,  l g   ⁄   mL) against selected Gram-positive organ-isms, viz.  Bacillus subtilis   (NCIM-2063),  Bacillus cereus   (NCIM-2156)and  Staphylococcus aureus   (NCIM-2079) and Gram-negative organ-ism viz.,  E. coli   (NCIM-2065) by the broth dilution method as recom-mended by the National Committee for Clinical LaboratoryStandards with minor modifications (22). Levofloxacin was used asstandard antibacterial agent. Solutions of the test compounds andreference drug were prepared in dimethyl sulfoxide (DMSO) at con-centrations of 100, 50, 25, 12.5, 6.25, 3.125  l g   ⁄   mL. Eight tubeswere prepared in duplicate with the second set being used as MICreference controls (16–24 h visual). After sample preparation, thecontrols were placed in a 37   C incubator and observed for anymacroscopic growth (clear or turbid) the next day.Into each tube, 0.8 mL of nutrient broth was pipette (tubes 2–7),tube 1 (negative control) received 1.0 mL of nutrient broth and tube8 (positive control) received 0.9 mL of nutrient. Tube 1, the negativecontrol, did not contain bacteria or antibiotic. The positive control,tube 8 contained bacteria, but not antibiotic. The test compoundwere dissolved in DMSO (100  l g   ⁄   mL), 0.1 mL of increasing concen-tration of the prepared test compounds which are serially dilutedfrom tube 2 to tube 7 from highest (100  l g   ⁄   mL) to lowest(3.125  l g   ⁄   mL) concentration (tube 2–7 containing 100, 50, 25, 12.5,6.25, 3.125  l g   ⁄   mL). After this process, each tube was inoculatedwith 0.1 mL of the bacterial suspension whose concentration corre-sponded to 0.5 McFarland scale (9  ·  10 8 cells   ⁄   mL), and each tubewas incubated at 37   C for 24 h at 150 rpm. The incubation cham-ber was kept humid. At the end of the incubation period, MIC val-ues were recorded as the lowest concentration of the substancethat gave no visible turbidity, i.e., no growth of inoculated bacteria.Results are presented in Table 1. Disc diffusion The inoculum can be prepared by making a direct broth or salinesuspension of isolated colonies of the same strain from 18 to 24 hMeller-Hinton agar plate. The suspension is adjusted to match the0.5 McFarland turbidity standard, using saline and a vortex mixer.Optimally, within 15 min after adjusting the turbidity of the inocu-lum suspension, a sterile cotton swab is dipped into the adjustedsuspension and then the dried surface of an agar plate is inocu-lated by streaking the swab over the entire sterile agar surface.Any surface moisture to be absorbed before applying the drugimpregnated disk (23).The plates containing bacterial inoculums are received a disc of lev-ofloxacin (20  l g) and synthesized compound (20  l g), whereas thecontrol plate was inoculated with DMSO which shows no inhibition Table 1:  Antibacterial activity of hybrid derivatives CompoundMinimum Inhibitory Concentration (MIC, in l g  ⁄   mL)Inhibition halo (in mm) Percentage inhibitionGram positive Gram negative Gram positive Gram negative Gram positive Gram negative B. subtilis B. cereus S. aureus E. coli B. subtilis B. cereus S. aureus E. coli B. subtilis B. cereus S. aureus E. coli  8a  25 100 6.25 25 24 09 36 24 54.5 22.5 85.7 52.2 8b  12.5 100 25 25 32 18 29 27 72.7 45 69.0 58.6 8c  6.25 25 25 12.5 38 28 27 32 86.3 70 64.2 69.5 8d  25 100 3.125 25 29 14 38 26 65.9 35 90.4 56 8e  25 12.5 6.25 50 27 34 35 22 61.3 85 83 47.8 8f  6.25 12.5 12.5 12.5 35 30 31 32 79.5 75 73.8 69.5 Levofloxacin  3.125 3.125 3.125 3.125 44 40 42 46 100 100 100 100 Design, Synthesis, Antibacterial Activity, and Molecular Docking Studies of 1,3,5-triazines Chem Biol Drug Des   2012  5
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